MEASUREMENT OF XYLANASE IN ANIMAL FEEDS

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1 MEASUREMENT OF XYLANASE IN ANIMAL FEEDS using XYLAZYME AX TABLETS K-XYLS 10/05 (200 Assays per Kit) Megazyme International Ireland 2005

2 INTRODUCTION: Arabinoxylan is the major endosperm cell-wall polysaccharide of wheat and rye and is found in significant proportions in most cereal solutions and slurries of high viscosity, and in animal nutrition it reduces the rate of nutrient absorption from the gut. endo-β-d-xylanase (xylanase) is added to feeds to catalyse depolymerisation of this polysaccharide. It can be demonstrated that endo-cleavage by xylanase of just one bond per thousand in the arabinoxylan backbone can significantly remove viscosity properties. Of the carbohydrase enzymes used as feed supplements, one of the most difficult to measure has been xylanase. These problems are attributed to several factors, including the low levels of enzyme added to the feed, inactivation of enzyme during pelleting, binding of the enzyme to feed components and the presence of specific xylanase inhibitors. The only biochemical methods which are sufficiently sensitive, specific and robust to measure xylanase in feeds are viscometric assays and those employing dyed xylan or arabinoxylan polysaccharides. Viscometric assays are tedious, whereas assays employing dyed xylan substrates are rapid, reproducible and simple to perform. We recommend the use of either Xylazyme AX tablets or Azo- Wheat Arabinoxylan (Azo-WAX). Xylazyme AX based assays are about 5-fold more sensitive than assays employing Azo-WAX. However, this latter substrate does have sufficient sensitivity in most applications, and results are slightly more reproducible than with Xylazyme AX. It is generally accepted that xylanase enzymes which are best suited to feed applications have optimal activity at ph 6.0. Consequently, these enzymes are generally assayed at this ph in 100 mm sodium phosphate buffer. In recovery experiments, however, we found that sodium phosphate buffer extracts only a small proportion (< 20%) of the amount of enzyme added to the feed. Thus a wide range of alternative extractants and extraction conditions have been evaluated. For feeds containing Trichoderma sp. xylanases, the best and most consistent results have been obtained using 100 mm acetic acid or 100 mm sodium acetate buffer (ph 4.7) at room temperature. Optimal extraction of Humicola sp. xylanases was achieved with a buffer containing 100 mm MES buffer (ph 6.0) and 1 % w/v sodium dodecyl sulphate (SDS). 1

3 KITS: Kits containing the required reagents to measure xylanase in animal feeds are available from Megazyme. These kits contain: 1. Xylazyme AX test tablets (200 tablets). 2. A. niger control xylanase (~ 295 mu/ml at 40 C and ph 4.7) in 50 % (v/v) glycerol (activity stated on vial). 3. T. longibrachiatum control xylanase (~ 386 mu/ml at 40 C and ph 6.0) in 50% (v/v) glycerol (activity stated on vial). EXTRACTION BUFFERS: (not enclosed): (A) Acetic acid (0.1 M) Add 5.8 ml of glacial acetic acid (1.05 g/ml) to 900 ml of distilled water and adjust the volume to 1 litre. Stable at room temperature for > 12 months. (B) MES buffer (100 mm) plus SDS (1 % w/v) Add 19.5 g of MES free acid (Sigma M-8250) to 800 ml of distilled water and dissolve. Adjust the ph to 6.0 with 1 M sodium hydroxide. Add 10 g of sodium lauryl sulphate (SDS; Sigma L-4509) and dissolve. Adjust the volume to 1 litre and add 0.2 g of sodium azide and dissolve. Stable at room temperature for > 12 months. EQUIPMENT (Recommended): 1. Glass test tubes (round bottomed; 16 x 100 mm and 16 x 120 mm). 2. Micro-pipettors e.g.: Gilson Pipetman 200 μl and 500 μl. 3. Positive displacement pipettor e.g.: Eppendorf Multipette - with 5.0 ml Combitip [to dispense 0.2 ml aliquots of xylanase control in 50% (v/v) glycerol]. 4. Adjustable volume dispenser set at 5.0 ml (to dispense Trizma base solution). 5. Top-pan balance correct to 0.01 g. 6. Spectrophotometer set at 590 nm. 7. Vortex mixer (e.g. IKA Yellowline Test Tube Shaker TTS2). 8. Whatman No. 1 (9 cm) filter circles and filter funnels. 2

4 EXTRACTION AND ASSAY OF XYLANASE IN FEED SAMPLES: Trichoderma sp. Xylanases: EXTRACTION: 1. Mill feed samples (approx. 50 g) to pass a 0.5 mm screen and mix thoroughly. 2. Weigh 0.5 g (± 0.01 g) of each sample in quadruplicate into glass test-tubes (16 x 120 mm). 3. Add 5 ml of 0.1 M acetic acid to each sample and stir on a vortex mixer. Add 0.2 ml of distilled to two of these tubes with stirring. To the other two tubes add 0.2 ml of control xylanase solution (approx mu/0.2 ml; see vial label) with vigorous and immediate stirring on a vortex mixer. 4. Incubate the slurries at room temperature and stir occasionally over the following 20 min. 5. Centrifuge the tubes at 1,500 g for 10 min in a bench centrifuge and use the supernatant directly in the assays. Assays should be initiated within 30 min of obtaining these extracts to minimise loss of enzyme activity in the extracts. ASSAY: 1. Accurately transfer 0.5 ml aliquots of supernatant solutions (in duplicate) to glass test-tubes (16 x 100 mm) at room temperature. 2. Add a Xylazyme AX tablet (without stirring) to each tube and immediately place the tubes in a water bath set at 50 ± 0.1 C and incubate for exactly 30 minutes. 3. After exactly 30 minutes, add 5 ml of Trizma Base solution (ph ~ 9), stir vigorously on a vortex mixer and store at room temperature for 5 minutes. NOTE: 1. This treatment terminates the reaction. 2. The tubes must be stored at room temperature and not at 50 C, as the substrate is not stable under alkaline conditions at elevated temperatures (i.e.: absorbance values will increase due to substrate breakdown). 3

5 4. Stir the tubes on a vortex mixer and filter the slurry through a Whatman No. 1 (9 cm) filter paper. 5. Measure the absorbance of the filtrates at 590 nm against a Reaction Blank. Prepare the Reaction Blank by adding Trizma Base solution (5 ml) to the feed extract (0.5 ml), followed by a Xylazyme AX tablet. Stir the slurry and store at room temperature for 5 min before filtration through Whatman No. 1 filter paper. A single Reaction Blank is required for each feed sample. CALCULATION OF ACTIVITY: The level of xylanase in the flour sample is calculated as follows: Activity in feed sample (0.5 g) = Added activity SA TA - SA where: Added activity = the amount of xylanase added to the feed sample slurry at the time of assay e.g.: 70 mu in the control xylanase solution (0.2 ml). SA = the reaction absorbance obtained for extracts of the feed sample to which no control xylanase was added. TA = the total absorbance i.e. the absorbance of extracts of the feed sample to which the control xylanase was added. EXAMPLE CALCULATION: Sample Abs/30 min. incubation 1. Feed A Feed A containing Trichoderma sp. xylanase (SA) SA + 70 mu xylanase (in the assay) Activity in feed sample (0.5g) = Added activity x SA TA - SA 4

6 where: SA = absorbance of extract of the sample [assayed by the standard format (e.g.: 0.859)] TA = total absorbance; i.e. the absorbance of extracts of the sample to which the additional xylanase (0.2 ml of 350 mu/ml) was added (e.g.: Abs = 1.299) Thus: Activity in feed (U/0.5g) = 70/1000 Units x 0.859/( ) = 0.07 x 0.859/0.440 = U/0.5 grams = x 2000 = 274 U/Kg or 274,000 Units/ton NOTE: Through the equation, the activity calculated is at 40 C and the ph at which the particular enzyme was standardised e.g. A. niger xylanase at ph 4.7 and T. longibrachiatum xylanase at ph 6.0. REFERENCE: McCleary, B.V. Problems in the measurement of β-xylanase, β-glucanase and α-amylase in feed enzymes and animal feeds. In Proceedings of Second European Symposium on Feed Enzymes (W. van Hartingsveldt, M. Hessing, J.P. van der Lugt and W.A.C. Somers Eds.), Noordwijkerhout, Netherlands, October, APPENDIX: Information on the percentage recovery of Trichoderma sp. xylanases added to feeds was obtained by performing incubations and assays under the standard conditions for feeds, with four levels of added enzyme, in the presence and absence of feed in the extraction mixture. The pelleted feed which was used was milled (< 0.5mm) before use. The recovery of activity was approximately % of the added activity (Figure 1). The line obtained for enzyme without added feed is curved, whereas the line for enzyme recovered from enzyme/feed mixtures is linear. In Figure 2, results are shown for a feed sample which had been sprayed with enzyme after pelleting and for a sample of the same feed which had not been sprayed with enzyme. It is apparent that recovery of enzyme added at the time of extraction is linear and that the curves for the two feeds are approximately parallel. 5

7 B A Figure 1: Curves showing the recovery of T. longibrachiatum (pi 9.0) xylanase (as absorbance 590 nm on hydrolysis of Xylazyme AX) from feed/enzyme mixtures (A), in comparison to measured activity in preparations free of added feed (B). A B Figure 2: Effect of enzyme added at the time of assay on the measured absorbance values. The two feed samples analysed were identical except that one was sprayed with enzyme post-pelleting (A) and the other was not (B). 6

8 The observation that the best extractant for T. longibrachiatum xylanase was 100 mm acetic acid was quite surprising. Under these extraction conditions (0.5 g feed per 5 ml of 0.1 M acetic acid), a final extraction ph of is obtained, whereas the optimal ph for activity of this enzyme is 6.0 (with only 70 % of maximal activity at ph 4.9; refer to Figure 3). This result suggests that the extractant either selectively solubilises a particular form of the enzyme (i.e. the pi 5.5 form which has a ph optima of 4.0) or that it extracts some other component which associates with the xylanase resulting in a change in ph activity characteristics. The first possibility has been discounted based on the observations that: 1. the pi 5.5 form of the enzyme represents only a small proportion of the total xylanase in the mixture which was evaluated, and 2. when the pi 5.5 and 9.0 forms of T. longibrachiatum xylanases were separated, purified and evaluated in binding studies, it was found that the pi 5.5 form binds to the feed more strongly (lower recoveries; see Figure 4) than does the pi 9.0 form. A B Figure 3. ph activity curves for the original T. longibrachiatum enzyme preparation (A) and for the xylanase fraction extracted from the feed sample (B). 7

9 A B C Figure 4. Binding of different highly purified xylanase enzymes to feed components. Extraction and assay conditions are as described in the text. A. A.niger xylanase; B. T. longibrachiatum xylanase pi 9.0 form C. T. longibrachiatum xylanase pi 5.5 form. NOTES: 8

10 NOTES: 9

11 NOTES: 10

12 Megazyme International Ireland, Bray Business Park, Bray, Co. Wicklow, IRELAND Telephone: (353.1) Facsimile: (353.1) Internet: WITHOUT GUARANTEE The information contained in this booklet is, to the best of our knowledge, true and accurate, but since the conditions of use are beyond our control, no warranty is given or is implied in respect of any recommendation or suggestions which may be made or that any use will not infringe any patents. 11

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